This invention relates to an improved particle gun for transferring biological materials such as nucleic acids into the cytoplasm of living cells. With the rapid advancement of recombinant DNA technology, there is a wide-ranging need for biologists to transfer biologic substances from one cell to another, and to transfer synthetic biological material into living cells to exert their activity therein. Such materials can include biological stains, proteins (antibodies or enzymes), and, most commonly, nucleic acids genetic material (either RNA or DNA). Most of the techniques used are painstakingly slow and use methods which transport materials into, at most, only a few cells at a time. More recently, there has been developed a particle bombardment process which utilizes a particle gun, as described in Sanford, et al, 1987, "Delivery of Substances Into Cells And Tissues Using A Particle Bombardment Process," Journal of Particle Science and Technology 5:27-37, the disclosures of which are hereby incorporated herein by reference. The basic particle gun disclosed in this article is illustrated at FIG. 3 of page 33 of the article. There it can be seen that the particle gun comprises a firing pin, barrel, a macroprojectile and a stopping plate, surrounded by a vacuum chamber. The barrel of the Sanford et al. gun has a smooth bore. In using a particle gun to transfer biological material into the cytoplasm of living target cells, it is important to avoid carrying unwanted debris into the cells which might result in damage or death to a large number of cells. Debris is undesirable because it may interfere with the success of the procedure; in fact it may cause the target cells to die. Unwanted cellular damage referred to as "blow by" results from debris that occurs when the macroprojectile is too small for the smooth bore barrel, or may result when the macroprojectile or stopping plate materials fail. Failure of the macroprojectile results in small particles of the ultra high molecular weight polyethylene (UHMWPE) material typically used extruding through the stopping plate at a high velocity. Stopping plate failure results when small pieces of the polycarbonate stopping plate are released from the plate upon impact by the macroprojectile. Any one of these sources of debris can substantially lower efficiency and increase unwanted cellular damage or death. The prior art particle gun has a vent near the forward muzzle end of the barrel. However, this venting, even when used with an associated vacuum, is unsuccessful in preventing blow by.
Another problem with the earlier-described Sanford et al. particle gun is poor tolerance for macroprojectile size variations within the range normally obtained by conventional manufacturing methods, whether machining or extrusion. Since the macroprojectiles manufactured from UHMWPE vary in size by as much as 0.002 inches as they are delivered, many of these must be discarded.
Accordingly, one objective of the present invention is to provide an improved particle gun which produces an increased number of transformed cells while at the same time lessening the risk of cell damage or death. Another objective of the present invention is to further increase the number of transformed cells by employing inert high mass metallic beads as microprojectiles in combination with certain preferred polyamine adjuvant materials.
The method and means of accomplishing each of these objectives will become apparent from the detailed description of the invention which follows hereinafter.